Elucidation of the genetic, hormonal, and cytophysiologic factors regulating the expression of phenylalanine hydroxylase (PHXase) activity in rat-hepatoma cells in culture. The levels of PHXase measured in particle-free extracts of cultured rat-hepatoma cells are being studied as a function of cellular hormonal environment, cytosocial behavior (i.e., stage in the population-growth cycle of the cells), and position in the mitotic cycle. Attempts will be made to establish novel cell lines from both normal and neoplastic human and rodent liver tissue in order to compare the regulation of this differentiated function in normal and neoplastic tissues. Furthermore, the comparison of the cytosocial behavior and cell-cycle kinetics of established cell lines from liver and hepatoma implicit in this work may provide additional insight into the differences in these characteristics between normal and neoplastic cells. (1) Two established cell lines derived from transplantable rat hepatomas (H4-II-E-C3 and MH1C1) express physiologic levels of PHXase. Maximum levels of the enzyme require the presence of both glucocorticoid hormone and as-yet-undefined nonsteroidal factors present in mammalian sera. (3) Immunologic and kinetic criteria indicate that the enzymes from the hepatoma cells and from adult-rat liver are very similar, if not identical. (4) Maximum sensitivity to stimulation of cellular PHXase by glucocorticoids and by serum is found only after cultures attain confluency. (5) A tyrosine-free medium for the selective growth of cells expressing PHXase activity out of a phenotypically mixed population has been formulated and tested; this selective medium should greatly facilitate the establishment of novel cell lines from normal and neoplastic rodent and human liver tissues.